Potassium channel activity in a cell is a critical determinant of normal cell function. This activity depends on an ensemble of channel properties including permeation and gating. Permeant ions themselves modulate these properties, thereby suggesting a potential means of autoregulation of channel activity, which could be important for homeostatic electrical activity. Our long term goal is to understand the autoregulatory mechanisms by which permeant ions modulate and synergize pore properties, gating (specifically slow inactivation), and movement of the voltage sensor. This proposal is divided into two aims. The first aim investigates the mechanisms of permeant ion modulation of gating and permeation in potassium channels. Several hypotheses will be considered by exploiting a series of Shaker mutants having different kinetics of slow inactivation. Specifically, we will test how mutants that enhance the rate of slow inactivation have drastically reduced macroscopic conductances to Cs + ions, whether this is due to altered permeabilities/selectivity, lower values of single channel conductance, altered ion occupancy of the pore, and/or decreased probabilities of opening. Among other electrophysiological approaches, we will use nonstationary noise analysis of currents expressed in HEK cells. The second aim will test whether ions in the selectivity filter modulate the movement of voltage sensors. These studies will entail measurements of kinetics and voltage dependence of gating currents in HEK cells.